Saccharide libraries

ABSTRACT

Novel methodologies for producing saccharide libraries are provided as well as the libraries themselves.

The invention relates to the production and functionalisation of heparansulfate sequences and related sequences. The invention finds applicationin the production of heparan sulfate and related sequences, diverse andfocused libraries of such sequences and the determination of functionsassociated with the sequences.

Heparan sulfate (HS) proteoglycans are cell-surface molecules widelyfound on mammalian cells and consist of a core protein and complex,sulfated linear glycosaminoglycan (carbohydrate) chains. These sugarchains belong to the wider glycosaminoglycan (GAG) family, which alsocontains chondroitin sulfate, dermatan sulfate and keratan sulfate. HSchains bind to a variety of molecules including growth factors, enzymes,adhesion molecules and receptors and it is these interactions that arethought to underlie the large number of biological activities attributedto HS. Heparan sulfate consists of linear polysaccharide chains composedof repeating glucosamine-glucuronate and glucosamine-iduronatedisaccharides. These saccharides can be modified by attachment ofcertain chemical groups at various, but restricted, positions to thesaccharide rings. Glucosamine (sometimes designated A-standing foraminosugar) can possess an N-sulfate or N-acetyl group attached to thenitrogen atom (N-) and O-sulfates at position 6 or, more rarely, 3 (6-O,3-O sulfates). Iduronate (sometimes designated I) can frequently, andglucuronate (sometimes designated G) more rarely, possess sulfate atposition 2 (2-O sulfate). A combination of these structures within thenaturally occurring heparan sulfate allows the creation of chainscontaining diverse, unique sequences of saccharides.

Heparan sulfate is structurally the most complex of the GAGs, both interms of the variety of its constituent monosaccharides and thecomplexity of their arrangement along the sugar chain (i.e thesequence). Particular HS saccharide sequences (displaying particularpatterns of sulfation) bind to specific proteins and these HS-proteininteractions underlie a huge variety of cellular functions (includingdevelopment, differentiation, growth and repair mechanisms) and alsomany disease processes (eg. heart and blood vessel disorders, cancer,asthma, arthritis, Alzheimers). Many reports in the literature alsosuggest that interactions between HS on the surface of mammalian hostcells and a wide range of pathogens and parasites are important forinfection. The interactions between individual HS sequences and proteinsor cells are therefore major new therapeutic targets. The identificationof bioactive HS sequences and the characterisation of the mechanism oftheir action will permit compounds which mimic or block sugar functionsto be discovered, potentially leading to a new class of drugs targettinga range of diseases. Indeed, the identified sequences themselves havepotential as novel drugs.

One major practical problem in this area of research is the scarcity ofHS available from natural sources. A commonly used approach is to employthe widely available, structurally related, but generally more heavilysulfated molecule heparin, which is itself a very widely-usedantithrombotic agent. Heparin, which shares the same underlyingstructural framework as HS, is considered by some to be a form of HS andexhibits a range of compositions dependent on its origin. However, itpossesses higher overall levels of sulfation and, generally, contains alower proportion of glucuronic acid and N-acetyl glucosamine residues.While these properties have sometimes lead heparin to be considered as amore homogeneous compound than HS, it is nevertheless, still considereda relatively complex molecule.

Previous work in this area has attempted to simplify this relativestructural complexity of heparin because it was considered acomplicating factor. Indeed, HS, heparin and oligosaccharides derivedfrom them have frequently been considered as intractable for structuralstudies precisely because of their sequence complexity. This isparticularly so when mixtures of saccharides are produced because theycan contain large numbers of structures, often similar or related, thatare difficult to separate. In response to this complexity, attempts havebeen made to instigate simple global changes, for example, by removingall of one particular type of sulfate group and observing how thischange influences the activity of the sample. The intended result ofsuch work is to make the correlation of biological activity andstructure more straightforward. The individual chemical processes havecomprised:

Selective de-sulfation of N-sulfated glucosamine

De O-sulfation in iduronate and glucosamine residues

Selective de-O-sulfation of iduronate residues

Selective de O-sulfation in glucosamine residues

N-sulfation of unsubstituted amino groups in glucosamine

N-acetylation of unsubstituted amino groups in glucosamine

There are many examples of this type of approach in the scientificliterature and a typical example of the overall philosophy is outlinedin Kariya et al J. Biol. Chem. (2000) 275 25949-25958 who stated:“Specific removal of major sulfate groups of heparin such as2-O-sulfate, 6-O-sulfate, and N-sulfate groups would be useful in orderto clarify the backbone structures of oligosaccharides bearing specificarrays of sulfate groups responsible for the interactions withphysiologically active molecules. For instance, selective removal of6-O-sulfate groups from glucosamine residues of heparin is of greatimportance in order to evaluate the involvement of 6-O-sulfate group(s)in the interaction between heparin, bFGF, and FGF receptors (FGFRs).”

This prior art approach has been broadened to include work in which thechemical steps have been carried out to completion in several positions.The object in these cases has still been to make products that arestructurally simpler than the starting material, again, with the aim ofcorrelating biological activity and structure. The Inventors havepublished similar work in which combinations of complete (and onepartial) modifications and their structural characterisation by ¹H and¹³C NMR were described and a more recent publication in which singlecomplete, as well as the combination of a complete and a partialchemical modification to heparin, have been correlated with biologicalactivity. However, such an approach can be criticised on the groundsthat it is not necessarily the case that chemical groups can be removedpiecemeal without affecting the structure of the molecule in some otherway.

By taking an alternative approach, the Inventors have devised themethods described herein, which can deliberately create libraries ofcompounds derived from heparin/HS that increase still further thestructural diversity within the HS sample and, indeed, have thepotential to create maximum structural (and hence sequence) diversitypossible within the limits imposed by the nature of the material (i.e.heparin/HS) and the chemistry of the individual steps, whilst includingsubstitutions only at those positions of the constituent monosaccharidesthat are found substituted in the naturally occurring products. Indistinction to the prior art, in which efforts concentrated on findingbiologically relevant structures within heparin or HS, the Inventors areinterested in finding optimised active structures from the vastlyincreased pool of structures available by this approach, irrespective ofthe representation of such structures within the naturally occurringproducts.

In addition, the processes described herein are distinct from manyexamples in the prior art, in which HS (and GAGs in general) have beenmodified in ways additional to removal of sulfate groups from positionsfound sulfated or acetylated in the naturally occurring material. Forexample, those methods in which sulfates are introduced at position 3 ofiduronate or O-acetyl groups are introduced. In the current invention,no sulfate or acetyl group is added to positions within the constituentmonosaccharides that is not found to bear this group in themonosaccharide units contained within the naturally occurring material.

Several examples of apparent chemical modification to heparin, HS orrelated GAGs can be found in the patent literature, for example, U.S.Pat. No. 5,430,133, U.S. Pat. No. 5,405,949, U.S. Pat. No. 5,543,403,U.S. Pat. No. 5,958,899, U.S. Pat. No. 4,717,719 and EP0380719. Inparticular, selective de-O-sulfation at iduronate-2-sulfate groupsemploying highly basic conditions. This modification is intended toresult in selective removal of 2-O-sulfate groups from iduronate; infact, it also results in the introduction of unnatural modifications (inthe small amounts of N,3 disulfated and N,3,6 trisulfated glucosamineresidues present in heparin, see Yates et al., Carbohydr. Res., (1997)298 335-340) while its incomplete application introduces epoxide groupsin the iduronate residues (see M. Jaseja et al., Can. J. Chem., (1989)67 1449-1456). The present invention does not rely on the introductionof any such abberant substitutions.

Furthermore, the compound libraries produced by the methods of thepresent invention have the capacity to be “tuned”, i.e. the methods canbe used to find an active compound or one minimising, for example, sizeand charge, and then regenerate a sub-library of related, but subtlydifferent structures, some of which may exhibit improved activity. Thisallows a chosen property of these molecules to be optimised, for examplesize, charge or activity, and further compounds to be produced in whichthe chosen property is enhanced.

Thus in a first aspect, the invention provides a method for theproduction of a library of heparan sulfate derivatives produced by acombination of chemical modifications selected from the group A to O:

-   A. partial de N-sulfation in glucosamine-   B. complete de N-sulfation in glucosamine-   C. partial de N-acetylation in glucosamine-   D. complete de N-acetylation in glucosamine-   E. re N-sulfation in glucosamine of all available amino groups-   F. re N-acetylation in glucosamine of all available amino groups-   G. partial re N-sulfation in glucosamine-   H. partial re N-acetylation in glucosamine-   I. complete de-O-sulfation at position 6 of glucosamine-   J. partial de-O-sulfation at position 6 of glucosamine-   K. partial de-O-sulfation at both position 6 of glucosamine and 2 of    iduronate accompanied by complete de N-sulfation in glucosamine.-   L. complete de-O-sulfation at both position 6 of glucosamine, 2 of    iduronate and de-N-sulfation in glucosamine-   M. partial de-O-sulfation at position 6 and complete de-N-sulfation    of glucosamine-   N. complete de-O-sulfation at position 2 of iduronate-   O. complete de-O-sulfation at position 6 and de N-sulfation of    glucosamine and partial de-O-sulfation of iduronate

Partial means not all of the available groups are modified, completemeans all of the available groups are modified.

Whilst it will be understood that two or more compounds can constitute alibrary, the methods of the invention allow libraries to be made inwhich structural diversity is increased compared to the startingmaterial (HS/heparin), or used to their ultimate extension, structuraldiversity is maximised, i.e. combinations of modifications are chosensuch that the library contains HS molecules with very highly diversechemical structures. Libraries produced by the methods of the inventionalso permit re-preparation of the components or for their production tobe optimised, that is, to be tuned towards compound(s) with desiredstructures and/or functions (or new, but structurally related ones to bemade). Such compounds may possess minimum size or charge but retain acertain level of activity, for instance. The methods of the inventionallow the deliberate increase of structural diversity (i.eheterogeneity) in compound libraries. One method of ascertaining theoverall level of structural diversity present in such samples is toconduct enzymatic (e.g heparatinase I, II and III) and/or chemicaldegradation and observe the pattern formed by the products on aseparative technique, for instance gel electropherogram or HPLC trace.

The term “heparan sulfate” is defined herein to include heparan sulfate,heparin, heparan sulfate-like GAGs or other heparin-like GAGs either inthe form of polysaccharides, often considered to be longer than 20monosaccharide units, or in the form of oligosaccharides, generallyconsidered in the art to comprise fewer than 20 monosaccharide unitsalthough the boundary between the two is essentially arbitrary. Someauthorities consider heparin to be a subclass of heparan sulfate, othersthat it is distinct. In any case, both are members of the widerglycosaminoglycan family. Herein, “heparan sulfate” also means anyderivative of the above list formed by combinations of modificationsfound in the prior art. Thus the methods of the invention may be used tofurther modify heparan sulfate derivatives made by methods other thanthose described herein. “heparan sulfate derivatives” means compoundsproduced from the methods of the invention, including the modificationsof heparin or heparan sulfate described herein and any further methodsteps, for example digestion of a modified polysaccharide, to produce apool of oligosaccharides, or other chemical modifications. “Heparin orheparan sulfate” used herein includes glycosaminoglycan moleculesderived from natural sources, or those arising from chemicalmodification of these compounds, or fragments, multivalent complexes oraggregations derived from these.

Various combinations and orders of modification reactions are logicallypossible in the methods of the invention. By “any combination” it ismeant all combinations or orders of modification steps except where thecombinations or orders are not considered logically possible by a personskilled in the art. By naming the position and type of sugar in which amodification is made, (for example, position 6 of glucosamine, orposition 2 of iduronate—also called glucosamine-6-O-sulfate oriduronate-2-O-sulfate respectively), it is meant that these changesoccur throughout the sample and to the extent indicated (partial orcomplete) and, in the case where a single species has not been isolated,it means that this property is that observed when averaged over thewhole sample. This will include a distribution of molecules withmodifications of different extent within the sample.

It will be clear to a person skilled in the art that variouscombinations of the modification steps are possible, including forexample:—

(i) Incomplete de N-sulfation in glucosamine; this can be achieved aloneor at the same time as de-O-sulfation (either partial or complete atposition 6 of glucosamine or 2 of iduronate).

(ii) Complete de N-sulfation; this can be achieved alone under mildconditions, as in (i) above, but also occurs under harsher conditionssuch as those used to achieve 6 de-O-sulfation in glucosamine,2-de-O-sulfation in iduronate and, under yet harsher conditions,complete de-O-sulfation throughout.

(iii) It is also possible to remove 2-O-sulfates in iduronate residuesselectively only if all of these are removed. This reaction takes placevia a different reaction to those above, but if it were carried out toonly partial extent, it would result in the formation of unwantedepoxide groups in some of the former iduronate-2-sulfate groups.

(iv) 6-O-desulfation of glucosamine can be achieved by reacting thepyridinium salt of heparin in pyridine with a silylating agent, MTSTFA(N-methyl-N-(trimethylsilyl)trifluoroacetamide), to form silylatedderivatives. These can then be selectively cleaved under aqueousconditions to give a derivative containing 6 de-O-sulfated glucosamineresidues either to partial or complete extent.

(v) De-N-acetylation in glucosamine by certain methods e.g. NaOH andheat results in the formation of epoxides and probably also de-sulfationin previously 2-O-sulfated iduronate residues. A similar method ofcarrying out de N-acetylation in glucsoamine involves treatment withhydrazine.

(vi) Selective re-N-sulfation and re-N-acetylation in glucosamine,either partial or complete, are easily achieved as described herein.

The predominant repeating disaccharide structure of heparin and heparansulfate can be shown as:

-   -   4) L-iduronic acid alpha(1-4) D-glucosamine alpha (1        where R₁=H, or O-sulfate (SO₃ ⁻), R₂=H, or O-sulfate and R₃=H,        or N-sulfate (SO₃ ⁻). Beta D-glucuronic acid and its        2-O-sulfated derivative can replace iduronate. Glucosamine can        be N-acetylated. In addition, there is a small amount of        glucosamine bearing 2,3 and 2,3,6 di and trisulfate groups.

The general structure of heparan sulfate (and heparin) is based on arepeating disaccharide composed of alpha (1-4) linked uronic acid(either alpha-L-iduronic acid or beta D-glucuronic acid) 1-4 linked toalpha-D-glucosamine to form a linear polysaccharide, which is thendecorated with a combination of O- and N-sulfates and/or N-acetyl andfree-amines. In the case of O-sulfates, these may occur at position-2 ofthe iduronate residue (and also more rarely at position-2 ofglucuronate) and position-6 of glucosamine (and occasionally atposition-3 of glucosamine). At the amino function of glucosamine,N-sulfate, N-acetyl and (it has been suggested) free amines can exist.Considering only the predominant repeating disaccharide of heparin; -4)alpha-L-iduronate (1-4) alpha-D-glucosamine (1-, There are twelvepossible theoretical combinations of substitutions (2 at iduronate-2:hydroxyl or O-sulfate, 2 at glucosamine-6; hydroxyl or O-sulfate and 3at glucosamine-N; free amine, N-sulfate or N-acetyl, giving 2×2×3=12combinations).

For a tetrasaccharide there are, therefore, 144 possible combinations(calculated from 12^(N/2), where N=the degree of polymerisation, hereN=4) and for a hexasaccharide. (N=6), there are 1728 combinations etc(i.e. these molecules contain a much higher degree of potentialdiversity than, say, peptides). Most of these sequences have not beenfound naturally occurring, but are nonetheless theoretical possibilitiesif the chemistry can be exploited. So, the relative complexity ofnaturally occurring HS is but a fraction of that possible if allsequence combinations are considered. Added to this level of sequencecomplexity is the variable chain length both within the naturallyoccurring polysaccharides, their chemically modifed derivatives and theproducts formed from them by degradative techniques.

“Complete modification” as defined herein refers to modificationscarried out on all of those positions available for that modification;“partial modification” as defined herein refers to modifications beingcarried out to fewer than the total available positions, i.e. incompletemodification. These definitions must be understood within the limit ofdetection of the technique used (i.e. of the actual experiment, not thetheoretical limit of the modification). For example, 90, 80, 70, 60% ofthe modification reaction HS substrate (by which is meant the percentageof particular residues within the chains, not the percentage of thechains) has been converted to product. The gross structural change mightbe measured, for example, by ¹³C NMR and, practically, this is able todistinguish between, for instance 90, 80, 70, 60% levels of substitutionbut not between say, 99 and 99.9%.

Chemical modifications which result in accidental remnants, may be takeninto account and considered as complete modifications provided that asignificant proportion of the product is present in the library or inthe next step of modification. So, a complete modification (for exampleN-sulfation) can be defined as either converting all amino groups toN-sulfates or all available free-amino groups (i.e. those notN-acetylated) to N-sulfates. Partial modifications (e.g. N-sulfation) isdefined as meaning converting some, but not all amino groups, oravailable amino groups to N-sulfate, for example only 10, 20, 30, 40,50, 60% of groups are converted in the product. However, while remnantsof unmodified groups may remain, it would be expected that, if carriedout as part of the common practice of attempting to simplifycorrelations between the structure and function, such products would, incases where unacceptable levels remained, be re-submitted to a repeat ofthe reaction in order to increase the levels of the desiredmodification. Under such circumstances, it would be counter-intuitivefor a person skilled in the art to submit a compound known to containsignificant levels of unmodified groups to subsequent steps,particularly if this was another partial modification or other partialmodifications.

If a single sample of the starting material is taken and is subjectedprogressively to a chemical modification, the sample will first containan increasingly varied range of sequences within the saccharide chains.If the treatment is continued, a maximum level of structuralheterogeneity will be reached but, as more and more of the individualdisaccharide units within the chains find themselves adjacent todisaccharides of identical structure, the sample will becomeprogressively homogeneous. This describes the situation within a singlesample along a simple reaction trajectory. A library of such compoundscould contain not only many compounds, for example, taken at variouspoints along this single reaction trajectory but, also many more takenalong a large number of different, single and multiple reactiontrajectories. The result is that libraries according to the inventioncan potentially possess huge diversity. For example, in a library of HSsaccharides of twenty monosaccharides, there are theoretically,12^(20/2)=12¹⁰ (in excess of ten thousand million) possible sequences.While it might be theoretically possible to access all of these,practically it is unlikely and, in any case, it would be impossible atthe present time to assess this number of structures. An importantpoint, however, is that such a library still allows a vast number ofpotentially active sequences to be uncovered that, hitherto, have beenneither found nor made.

In a preferred embodiment of the invention, a “library” of compoundscomprises at least 50 compounds

The degree of structural complexity within such a sample can bequalitatively assessed by monitoring its breakdown products by someseparative technique, (e.g. hplc or gel electrophoresis) following, forexample, heparitinase enzyme digestion or nitrous acid degradation. Thelevel of diversity within the library will depend on the number ofpoints at which samples have been taken during chemical modification andon the particular combinations and extents to which those modificationshave been taken.

Thus the invention provides methods for the creation of a library ofmodified heparan sulfate derivatives wherein said library isstructurally more diverse than the heparan starting material from whichit is derived.

Further examples of possible modifications are as follows;

(i) Selective de N-sulfation; either partial or complete and, by usingharsher conditions but the same reactants, complete de N-sulfation canbe accompanied by partial de-O-sulfation (either partially orcompletely) at position 2 of iduronate and position 6 of glucosamine.

(ii) Selective de-O-sulfation of iduronate 2-O-sulfate; this is achievedby a different reaction than that mentioned above and can only becarried out to completion. Partial reaction invariably results in thepresence of unnatural epoxide groups forming in the iduronate residue.

(iii) Selective de-O-sulfation at position 6 of glucosamine; this can becarried out to completion, in which case it is accompanied by somede-O-sulfation in iduronate and complete de-N-sulfation in glucosamine.Alternatively, a reaction with a higher degree of selectivity forde-O-sulfation of 6-O- over 2-O-positions than in reaction (ii) aboveand reportedly resulting in few, if any, other modifications occurringin the structure is available. This can be carried out either partiallyor completely.

(iv) Re N-sulfation; this can be achieved with complete selectivity,either partially or to completion.

(v) Re N-acetylation; this is possible either partially or tocompletion.

In the following, it should be understood that certain modificationse.g. partial de-O-sulfation of glucosamine can therefore be achieved bydifferent routes, either carrying out one modification at a time, orconcertedly.

Thus one embodiment of the first aspect of the invention providesmethods for the production of a library of modified heparan sulfatederivatives wherein said method comprises a combination of chemicalmodification steps in which at least one, two or three modificationsteps of said combination are selected from the group A to O.

In a further embodiment, the invention provides methods for theproduction of a library of modified heparan sulfate derivatives whereinall steps of said combination are chosen from the group A to O.

In a further embodiment, the invention provides methods for thegeneration of a library of modified heparan sulfate derivatives whereinat least one modification step in said combination is a partialmodification.

In another embodiment, the invention provides methods for the creationof a library of modified heparan sulfate derivatives wherein at leastone modification is carried out at the amino function (N—) ofglucosamine. In a preferred embodiment, at least one partialmodification is carried out at the amino function (N—) of glucosamine.

Another embodiment provides methods for the generation of a library ofmodified heparan sulfate derivatives wherein at least two modificationsteps in said combination are partial modifications.

An additional embodiment provides methods for the creation of a libraryof modified heparan sulfate derivatives wherein at least threemodification steps in said combination are partial modifications.

A further embodiment provides methods for the generation of a library ofmodified heparan sulfate derivatives wherein a first step ofmodification is chosen from A, B, C or D, such that wherein step A ischosen, optional subsequent steps are one or more of E, F, G, H, I, J,K, L, M, N, O or wherein step B is chosen, optional simultaneous orsubsequent steps are one or more of E, F, G, H, I, J, K, L, M, N, O inany combination;

An additional embodiment provides methods for the generation of alibrary of modified heparan sulfate derivatives wherein a second step ofmodification chosen from E, F, G, or H is performed upon the modifiedproducts of said first step.

A further embodiment provides methods for the creation of a library ofmodified heparan sulfate derivatives wherein a third step ofmodification chosen from A, B, C, D, E, F, G, H, I, J, K, L, M, N, O isperformed upon the modified products of said second step.

Another embodiment provides methods for the creation of a library ofmodified heparan sulfate derivatives wherein a fourth step ofmodification chosen from A, B, C, D, E, F, G, H, I, J, K, L, M, N, O isperformed upon the modified products of said third step.

An additional embodiment of the invention provides methods for thecreation of a library of modified heparan sulfate derivatives whereinthe combination of modifications is chosen from a first step and secondto fourth optional steps such that: Optional Optional Optional FirstStep Second Step Third Step Fourth Step B(+/−any of I to O) G F/HB(+/−any of I to O) H E/G B(+/−any of I to O) E B(+/−any of I to O) F AF +/−any of I to O E/G A H +/−any of I to O E/G

In a preferred embodiment, the invention provides methods for thecreation of a library of modified heparan sulfate derivatives whereinsaid first step modification is B (+/− any of I to O), said second stepmodification is H, and said third step modification is E or G.

Another preferred embodiment of the invention provides methods for thecreation of a library of modified heparan sulfate derivatives whereinsaid first step modification is B (+/− any of I to O), said second stepmodification is G, and said third step modification is F or H.

In another embodiment, the invention provides a method for the creationof a library containing at least two modified HS derivatives.

Heparin/HS polysaccharides can be cleaved into oligosaccharides ofdiffering sizes using endoglycosidases and/or by nitrous acid or freeradical degradation (e.g. using hydrogen peroxide) which cleave atdifferent positions along the chain. Heparin/HS poly- andoligosaccharides can be separated according to size and charge usingchromatography.

Thus in another embodiment of the invention, methods are providedwherein chemical or enzymatic degradation products of such componentsare created.

In another embodiment of the invention, methods are provided wherein aseries of chemical modification steps is carried out by taking aliquotsfrom a reaction vessel, or where the steps are carried out to differentextents in discrete locations.

The methods of the invention not only enable the production of diverselibraries of HS derivatives, but also permit such libraries to be“tuned” or optimised for a desired structural or functional featurefound amongst the members of the library. In other words, once a memberof a library produced by the methods of the invention has beenidentified as having a desired overall structure and/or particularstructural feature (e.g. degree of sulfation, sequence, content of aparticular monosaccharide residue etc) and/or a desired function, forexample, it tests positive in an assay for inducing cell motility, thenfurther libraries can be produced by adjusting the modifications to givea new library. This may be of closely related derivatives, i.e.focussing in on producing more derivatives that are structurally and/orfunctionally similar to the active derivative.

Thus in a second aspect, the invention provides a method which comprisesthe additional steps (singly or jointly) of;

(a)(i) determining at least one functional property of one or morecompounds;

(b)(i) making a further library via the method according to any one ofthe above methods wherein said modifications are chosen according to thefunctional determination or determinations made in step (a)(i); and/or;

(a)(ii) determining at least one structural feature of one or morecompounds;

(b)(ii) making a further library via the method according to any one ofthe above methods, wherein said modifications are chosen according tothe structural determination or determinations made in step (a)(ii);and/or,

(b)(iii) making a further library via the method according to any onethe above methods, wherein said modifications are chosen according toboth said functional determination(s) made in step (a)(i) and saidstructural determination(s) made in step (a)(ii).

As defined herein, determining a structural feature means ascertainingany physical property that can be influenced or controlled by theprocesses described in the first aspect of the invention. Suchproperties are primarily position and extent of modification, forexample; iduronate-2 sulfate, glucosamine-6-O-sulfate and eitherN-sulfate, N-acetyl or free-amine in glucosamine residues and also thedimensions of the saccharides. Another structural feature could be thecharge properties of the saccharides. The dimensions of the saccharidescould be determined by gel-based techniques, comparing to standardsand/or mass spectrometry. The position and extent of modification can bedetermined in a gross fashion; averaging over the whole sample by, forexample, NMR; in more detail, for example, by disaccharide compositionalanalysis or, in yet more detail; by carrying out sequencing, employingfor example, gel-based techniques and/or mass spectrometry.

As defined herein, determining a functional property means screening oneor more components of a library produced by the above methods for aparticular desired biological function, for example, binding to aspecific biological entity or exhibiting a biological activity such asthe ability to stimulate cell-proliferation, differentiation ormotility.

Thus, libraries according to the invention can give structural orfunctional cues which may be used to create further “tuned” libraries.Two basic ways of “tuning” libraries of the invention are envisaged. Thefirst, which can be termed “analytical” facilitates the production, inhigher abundance of a component or components, (or closely relatedvariants, some of which, it is hoped, possess improved activity), with agiven structure, or structural feature, from a library, once somethingis known about the structure. The second, which can be called“empirical”, can increase the abundance of a compound with desiredcharacteristics, and possibly, find closely related variants withimproved activity, without necessarily knowing anything about thestructure of the product.

For example, in the “analytical” method of tuning (see FIG. 1), havingmade a series of products, for example, several oligosaccharide poolsfrom several partially digested polysaccharides and having separatedthem, for example, by hplc, into their components (or mixtures of a few,structurally related oligosaccharides), the one or ones showing aparticular property (for example an activity of interest) is/areselected and analysed for structural composition (for example, by NMR,mass spec, disaccharide composition or sequencing) and the informationso obtained (for example, size, charge, degree of sulfation oracetylation at various positions) is used to adjust the subsequentpreparation of the products of a further library or libraries(polysaccharide and/or mixture of oligosaccharides) to give theparticular structure in greater abundance i.e. to increase thelikelihood of it being made, or to create related compounds, which maypossess higher activity. This process can usefully be repeated severaltimes. In “analytical tuning” at the level of polysaccharides, a set ofstructurally diverse, chemically modified polysaccharides is made,constituting a library. This is tested for some activity and theactivity correlates with a particular structural feature e.g. highlevels of N-acetylation. Polysaccharides are then made based aroundincreased levels of N-acetylation and these products re-tested and somefound which, in this hypothetical case, possess higher activities.

An “empirical” (see FIG. 2) method of tuning involves testing the sameset of products (for example, oligosaccharides) for activity and, havinglocated the one(s) of interest, slightly varying the conditions ofproduction (which are known) around those used to produce thatparticular set of products. (Note that some indication of physicalproperty e.g. degree of overall sulfation may however become apparentfor instance from the compound's elution position on an hplc trace).This will give a second set of products, which are themselves thenscreened for activity (this process could be repeated several times).The preparation of the particular product is thereby optimised withoutnecessarily having any knowledge of what it is; that could be addressedat a later stage.

In a further example of “empirical tuning” at the polysaccharide level,a set of compounds may be tested for a particular activity withoutknowledge of the structural features of the components of thepolysaccharides, but with a knowledge of the steps taken during theirpreparation, and a particular polysaccharide component may be selectedfor a particular activity. Polysaccharides are then prepared basedaround these conditions and tested for activity and some found topossess improved activity.

In both of these definitions the words “increase abundance” include themeaning “increase abundance in an absolute or in a relative way”; thiscovers the possibility that it may, under certain circumstances, beadvantageous to increase the abundance of one component over another,which is not necessarily the same as optimising for the production ofone particular component per se. (A more detailed description of thetuning process and pictorial representations are given in the Examplesbelow with reference to FIGS. 1 and 2).

The invention also provides a method of producing a supplementarylibrary of heparan sulfate derivatives comprising steps (singly orjointly) of;

(i) screening (i.e. testing) a library of heparan sulfate derivativesfor compounds which have particular structural and/or functionalcharacteristics,

(ii) determining at least one structural feature of the compounds havingsaid particular structural and/or functional characteristics, or

(iii) determining at least one functional property of the compoundshaving said particular structural and/or functional characteristics, or

(iv) determining at least one functional and one structural property ofthe compounds having said particular structural and/or functionalcharacteristics; steps (ii), (iii) and (iv) being followed by step

(v) making said further library via the methods of any one of the abovemethods wherein the modifications and number of modification steps arechosen according to the determinations of steps (ii), (iii) or (iv).

In another embodiment, the invention provides a method wherein at step(v) above, a single combination of modification steps is chosen in orderto reproduce only the compound(s) having said desired characteristics.

Other types of tuning, for example, optimising the ratio of twoactivities, or the ratio between an activity and some structuralproperty are variants of the above and are hence considered within thescope of the invention.

In an additional embodiment, the invention provides a method wherein twoactivities, or the ratio between some structural property or twostructural properties (e.g. size and charge) of components of thelibrary are optimised by either of the above mentioned analytical orempirical tuning methods.

In another embodiment, the invention provides a method wherein thelibrary of heparan sulfate or heparan sulfate derivatives is made by amethod according to any of the above claims.

(iv) determining at least one functional and one structural property ofthe compounds having said particular structural and/or functionalcharacteristics; steps (ii), (iii) and (iv) being followed by step

(v) making said further library via the methods of any one of the abovemethods wherein the modifications and number of modification steps arechosen according to the determination of steps (ii), (iii) or (iv).

In another embodiment, the invention provides a method wherein at step(v) above a single combination of modification steps is chosen in orderto reproduce only the compound or compounds having said desiredcharacteristics.

Other types of tuning, for example, optimising the ratio of twoactivities, or the ratio between an activity and some structuralproperty, or two structural properties (e.g. size and charge) arevariants of the above and are hence considered within the scope of theinvention.

In an additional embodiment, the invention provides a method wherein twoactivities, or the ratio between some structural property or propertiesof components of the library are optimised by either of the abovementioned analytical or empirical tuning methods.

In another embodiment, the invention provides a method wherein thelibrary of heparan sulfate or heparan sulfate derivatives is made by amethod of the first aspect of the invention.

In an additional embodiment, the invention provides a method wherein thestructural determination(s) made at step (ii) or (iv) above is/areprovided by the discreet known location, in a spatially separatedlibrary, of the compounds having said particular structural and/orfunctional characteristics.

Once a compound having a desired structure or function has been foundwithin a library made by the methods of the invention, additionalquantities can then be re-made either following a structural analysis,or from a knowledge of its reaction history, for example from records ofthe modifications carried out or, preferably, by virtue of the fact thatcompounds can be spatially located in accordance with the reactions towhich they have been subjected. In either case, re-synthesis can becarried out without necessarily knowing any structural information.

Thus in a further embodiment, methods are provided wherein components ofthe library of heparan sulfate derivatives are spatially located toallow one or more of them to be remade by virtue of the fact that thespatial location corresponds to the process which has been applied toproduce that component or components.

-   There are a wide range of screening methods and approaches known in    the art which can be employed to detect or measure a functional    property of a component or components of the libraries (for example,    Guimond, S. E. and Turnbull, J. E. (1999) Curr Biol. 9, 1343-1346.    Irie, A., Yates, E. A., Turnbull, J. E. and Holt, C. E. (2002).    Development. 129, 61-70. Kreuger, J., Salmivirta, M., Sturiale, L.,    Gimenez-Gallego, G. and Lindahl, U. (2001) J Biol Chem. 276,    30744-52. Nadkarni, V. D. and Linhardt, R. J. (1997) Biotechniques.    23, 382-5. Nadkarni, V. D., Pervin, A. and Linhardt, R. J. (1994)    Anal Biochem. 222, 59-67).

These include; spatially separated components of the library beingtested in any in vitro or in vivo assay, or firstly being bound, eithercovalently or non covalently, to a surface. An assay may determine anability to bind, an affinity or activity of a component of the libraryfor, or against, for example, a protein, another carbohydrate, cells,viruses or other biological or chemical entity.

In other words, the screening of components, or spatially separatedcomponents of the library, can be performed:

-   -   in crystals as complexes with proteins or peptides    -   in free solution in vitro experiments as well as in vivo; or    -   immobilised on one or more of the following;        -   a matrix        -   a resin        -   on beads (including magnetic)        -   on derivatised surfaces

Attachment to this variety of surfaces and supports may occur viacovalent binding or non-covalent attachment and may be in the form ofslides, wells, plates, beads, compact discs etc. Surfaces can be, forexample, polypropylene, polystyrene, gold, silica, ceramics or metal,nitrocellulose, PVDF, nylon or phosphocellulose. All of these can beemployed to bring a component of the library into the proximity of atest compound, in order for some functional property of the librarycomponent to be determined. Having identified components of the librarywith the desired function, their production can be repeated and thecomponents further separated for re-screening using the assay. Thelocation of components can correlate with the history of treatmentsemployed to create that particular component.

In a further embodiment of the first and second aspects of the inventionprovides a library in the form of modified heparan sulfate derivativesin which the compounds contained therein are spatially separated atdiscreet known locations. This facilitates rapid screening and tuning.

In another embodiment, the invention provides an array comprising asurface upon which are deposited each at spatially defined locations, acomponent, or components of a library of heparan sulfate derivativesmade by the methods of the invention.

In a further embodiment the invention provides an array comprising asurface upon which are deposited each at spatially defined locations atleast two heparan sulfate derivatives, (poly- or oligosaccharides)derived from said derivatives, produced by the methods of the inventiondescribed herein.

Thus in the method of the second aspect of the invention the functionaldetermination(s) made at step a(i) and/or structural determination(s)made at step a(ii) is/are provided by the discreet known location, in aspatially separated library, of the compounds having said particularstructural and/or functional characteristics.

Each position in the pattern of an array according to the invention cancontain, for example, either:

-   -   a sample of heparan sulfate derivative(s) or    -   a sample of heparan sulfate derivative(s) bound to an        interacting molecule (for example, a protein or small molecule).        The interacting molecule may itself interact with further        molecules    -   a sample of heparan sulfate derivative(s) bound to a synthetic        molecule (e.g. peptide, chemical compound) or    -   a sample of two or more different HS derivatives or HS        oligosaccharides

Preferably, the heparan sulfate derivative at each position issubstantially pure but in certain circumstances mixtures of several ormany different heparan sulfate derivatives can be present at eachposition in the pattern of an array. Thus initial bulk screening of setsof HS derivatives or HS oligosaccharides can be carried out on the arrayto determine those sets containing compounds of interest.

An array as defined herein is a spatially defined arrangement of heparansulfate derivatives in solution, or in a pattern on a surface. In thelatter case, the heparan sulfate derivatives are preferably attachedeither directly or indirectly via covalent or non-covalent bonds.

In a further embodiment, the invention provides a method of screening alibrary containing at least two heparan sulfate derivatives produced bythe methods of the first and second aspect of the invention comprisingthe steps of:

(a) bringing all or a portion of said library into contact or proximitywith a molecule, complex of molecules, cell or organism of interest,

(b) detecting an interaction between one or more compounds within saidlibrary and the molecule, complex of molecules, cell or organism ofinterest,

The screening of the libraries of the invention can give rise to usefulcompounds. Thus in a further embodiment, the invention provides use ofone or more HS derivatives made by the methods of the invention orcomponents of the same e.g. oligosaccharides, as enzyme substrates e.g.of sulphotransferases, as enzyme inhibitors e.g. of heparitinases, asepitopes to antibodies or phage display antibodies or libraries ofthese, as inhibitors of protein activity or ligands to proteins, or ascomponents of multi- or polyvalent inhibitors of adhesin attachment inmicroorganisms (viruses, bacteria, tropanosomes to mammalian cells).

Naturally occurring heparan sulfate is scarce. However it may besynthesised by the methods of the invention which can produce a samplewhich is indistinguishable by some structural, functional orphysico-chemical property from naturally occurring heparan sulfate.

Thus, in a further aspect, therefore the invention provides a method ofproviding heparan sulfate, where heparan sulfate means a polysaccharidethat is indistinguisable by some test of activity or structure or otherphysico-chemical property from naturally occurring heparan sulfate.

The invention will now be further described by the followingnon-limiting examples which refer to the accompanying figures in which:

FIG. 1 shows a schematic of an example of the Analytical Tuning Process,illustrated by production of an oligosaccharide, (about which somestructural detail is ascertained during the process) from a library ofpolysaccharides.

FIG. 2 shows a schematic of an example of the Empirical Tuning Process,illustrated by production of an oligosaccharide from a library ofpolysaccharides. No knowledge of the structure of the isolatedoligosaccharide product or initial polysaccharides is necessary—only thesynthetic history of the initial components of the polysaccharidelibrary.

FIG. 3 is graphical illustration of how different chemically modifiedheparin preparations will contain a range of structures with variedlevels of desulphation. The graph shows 3 different preparations eachwith a particular average level of desulphation for each of 2 differenttypes of sulfate group (A and B). The average level is denoted by thecentre of the circles. For example, preparation I is 20% desulfated atgroup A and 50% desulfated at group B; preparation II is 50%/50%desulfated and preparation III is 75%/75% desulfated. Note that althoughthese are the average level of desulphation for these preparations, theywill contain a range of structures with a variety of combinations oflower or higher levels of desulphation at each position. This resultsfrom two factors: the complex mixture of different sized molecules,possessing different sequences and the statistical distribution ofchemical modifications within the sample. These are represented by therange of variations encompassed by the circles centred on the averagedesulphation level points. In each case a particular area of “structurespace” is occupied. This is a simplified version with just 2modifications shown. In more complicated preparations additionalmodifications could take this representation of structure space to 3dimensions or more.

FIG. 4 is an illustration of how the tuning process works. Initial stepsare denoted by black arrows, the feedback process following initialselection of an active component, by dotted arrows

FIG. 5 is an illustration of the binding of a target (detected by aseries of antibodies, one being fluorescently labelled) to a componentof a library immobilised repetitively onto amino-derivatised glassslides at spatially discrete locations. Solvent without the librarycomponent present was spotted in between the rows of library componentsas a control. The upper and lower panels show regions of identicalslides where immobilisation was via conventional heating or microwaving,respectively.

FIG. 6 The generation of oligosaccharide library components from aheterogeneous polysaccharide starting material. Clockwise: Panel A;electrophoresis of a heparitinase II digestion of the heterogeneouspolysaccharide (P) compared to that of bovine lung heparin standard (S),which is comparatively homogeneous giving a characteristic ladder: PanelB; gel chromatography separation of digest (P) on Sephadex G-50 alsoshowing equivalent elution position of a standard DP 12 oligosaccharidepool from (S): Panel C; HPAEC separation (0-2 M NaCl, pH 7, 90 mins) ofthe fraction of (P) which elutes at the same position as a bovine lungheparin DP 12 standard: Panel D; electrophoresis profiles of 3 examplepeaks from the HPAEC trace, X, Y and Z, compared to the standard ladderderived from bovine lung heparin (S): Panel E; Disaccharidecompositional analysis of peaks X, Y and Z. Disaccharides: 1; UA-GlcNAc,2; UA-GlcNAc(6S), 3; UA-GlcNS, 4; UA-GlcNS(6S), 5; UA(2S)-GlcNS, 6;UA(2S)-GlcNS(6S), 7; UA(2S)-GlcNAc, 8; UA(2S)-GlcNAc(6S)

FIG. 7 The process of selecting active oligosaccharides, approachingminimum structural complexity, and capable of forming an activesignalling complex between FGF1 and receptor 2c. Heterogeneouspolysaccharide starting material was partially digested and the productsfractionated into oligosaccharide fractions A-O (in order of decreasinghydrodynamic volume) by GPC. Panel A; activity assay of FGF1/R2c in BaFcells with representative, sized oligosaccharide pools B, D and I ofincreasing hydrodynamic volume from the GPC separation of theheterogenous polysaccharide digestion. The activity of bovine lungheparin (polysaccharide) is also shown as a positive control. Panel B;from these fractions, the smallest active fraction (D) was furtherseparated by HPAEC into fractions a-t (in order of increasing anioniccharge) and tested. The activity of representative samples c, f, l and rare shown for signalling of FGF1/R2c. The activities of the parentoligosaccharide pool (D) and bovine lung heparin (BLH) are also shown.

EXAMPLES Example 1 Targetted Chemical Modification of Heparin; Making aLibrary with Varying Degrees of N-Sulfation and N-Acetylation

There are several possible routes to obtain partially N-sulfated,N-acetylated heparan sulfate derivatives;

(1) partial de N-sulfation (by solvolytic desulfation under mildconditions, acidic treatment with aqueous mineral or organic acids),then re N-acetylation (e.g. by acetic anhydride in basic aqueousconditions) to substitute all unsubstituted amino groups. I.e. step oneis controlled, step two not.

(2) complete de N-sulfation (by the reactions listed above but underharsher conditions of temperature or strength of acid used), thenpartial re N-acetylation (e.g. limiting the amount of acetic anhydride,reaction time or temperature), then re N-sulfate (by reaction of asulfate donor, trimethylamine sulfurtrioxide in basic aqueousconditions) all remaining unsubstituted amino groups. I.e. step 2 iscontrolled, steps 1 and 3 not.

(3) complete de N-sulfation followed by simultaneous re N-sulfation andacetylation in the same vessel. This is possible because both reactionsare carried out in saturated aqueous sodium bicarbonate solution.

(4) by a process involving de N-acetylation (hydrazinolysis or treatmentwith base), then re N-acetylation and/or N-sulfation as described in 1to 3 above.

A summary of the essential components of the reactions mentioned aboveis given below;

1. De N-Sulfation

Several methods. Mild acidic cleavage using dilute acids plus time orheat to control extent. One method is to use solvolytic de-sulfation,which includes the use of the pyridinium (or other similar salt of anorganic base) salt of heparin (or derivative) disolved, or suspended, ina mixture of DMSO and either water, methanol or other alcohol. Theextent of de-sulfation is controlled with a combination of temperatureand time. Other possibilities include heating in aqueous mineral ororganic acids. If conditions are mild, selective de N-sulfation can beachieved, either partially or to completion.

2. Re N-Acetylation

This can be achieved using acetic anhydride on a solution of the heparinor derivative in solutions of sodium bicarbonate or similar watersoluble base. It is usually carried out at low temperature followed byfurther reaction at room temperature; The extent of N-acetylation iscontrolled with the amount of reagent, temperature—or by the duration ofthe reaction.

3. Re N-Sulfation

This is achieved by using the trimethylamine.sulfurtrioxide complex (ora similar amine-sulfurtrioxide complex) on an aqueous solution ofheparin (or derivative) and sodium bicarbonate, or a similar watersoluble base. Characterisation of the products formed in these reactionscan be done by 1H and 13C NMR, degradation with enzymes or nitrous acid,followed by any separation technique or by elemental analysis (to findtotal sulfation), or titration (to find N and O sulfation ratios).

Example 2 Synthesis of a Library Component Containing Partial O,NSulfation and N-Acetylation from Heparin, Using the Chemical StepsDescribed Above

1. Preparation of Heparan Sulfate Derivative with Partial Ido-2De-Sulfation and Glucosamine-6 De-Sulfation

Heparin was converted to its pyridinium salt by passage through anacidic ion exchange column followed by neutralisation with pyridine andevaporation of excess water and pyridine to give the salt. This was thensuspended in DMSO/MeOH (9/1, v/v) and heated (e.g. 18 h, 65 degrees C.).The reaction was cooled, and the pH adjusted to 8 with dilute NaOH.Products were precipitated into a large volume of cold ethanol and theproducts precipitated. The products were recovered by filtration, saltslargely removed by dialysis and the products purified by desalting andthe product, heparin derivative A, characterised. This results in aproduct with completely de N-sulfated glucosamine residues and partiallyde-O-sulfated residues at position 2 of iduronate and 6 of glucosamine.

2. Preparation of Heparan Sulfate Derivative with Partial N-Acetylation

Heparin derivative A (100 mg) was dissolved in an aqueous, saturatedsolution of NaHCO₃ (5 ml) at 4 degrees C. and acetic anhydride (2.5molar equivalents) was added dropwise. The reaction was maintained at 4degrees C. for another 4 hours and then allowed to reach roomtemperature and stirred overnight. After completion of this reaction,the solution was poured into a large volume of cold ethanol and theproducts and salts precipitated. The products were recovered byfiltration, salts largely removed by dialysis and the products purifiedby desalting and the product characterised.

3. Re N-Sulfation of Remaining Unreacted Amino Groups.

The product of steps (1) and (2) was dissolved in a saturated aqueoussolution of sodium bicarbonate (10 ml) and a 10-fold molar excess oftrimethylamine sulfur trioxide complex was added, with stirring at 50degrees C. overnight. The reaction mixture was then cooled and thepolysaccharide products were precipitated into cold ethanol, filtered,dialysed, recovered and purified. The products were then characterised.

Example 3 Screening Components of the Library as an Array for Binding toTarget Proteins and Cells

Spatially separated components of a library were spotted in formamideonto glass slides possessing functional amino groups using a roboticspotter. The immobilisation reaction was allowed to proceed at 37 to 80°C. for at least 5 days. Alternatively, the slides were heated in aconventional microwave oven (850 W) at half power for five minutesbefore standing at ambient temperature in the dark for ten minutes andrepeating this procedure again twice. The arrays were then washed in asuitable solvent and incubated sequentially with bovine serum albumin(BSA), target (e.g. peptide, protein or cells) and then primary antibodyraised against the proteins or cells and secondary antibody (if eitherrequired) all diluted to appropriate concentrations in a suitablebuffer. The target, primary or secondary antibody are labelled with asuitable fluorophore for detection. At each step followingimmobilisation the slide was washed with a suitable solvent. After thefinal step the slide was washed with solvent, dried and scanned using afluorescent slide scanner producing a image such as FIG. 5

Example 4 Assaying the Components of a Library for the Ability toStimulate BaF3 Cell Proliferation

BaF3 cells are a pre-lymphoid cell line, lacking HS chains andexpressing a type of fibroblast growth factor receptor. BaF3 cells weretransferred at a suitable cell density from medium supplemented withinterleukin 3 growth factor (IL3), required as a survival factor, intomedium lacking IL3 and supplemented with a suitable concentration of afibroblast growth factor (FGF) and the component of the library undertest. As controls cells are also transferred to medium lacking both FGFand the library division as well as to medium possessing one of thesupplements alone. The cells were incubated at 37° C. with 5% carbondioxide for a suitable period of time before determining the number ofviable cells and comparing the library division results with thecontrols.

Example 5 Production of a Diverse Library; Its Use to Identify ActiveStructures, to Tune the Library for the Production of More ActiveFragments

(i). A sample of the starting material e.g heparin is taken

(ii). A number of modifications according to the first aspect of theinvention to cover the desired degree of structural diversity arecarried out

e.g. a graded series of N-acetylations in combination with a gradedseries of de-O-sulfations (the preferred route). This is done asfollows:

-   -   Some heparin is taken    -   Partial O-de sulfation and simultaneous complete de-N-sulfation        is carried out;    -   The pyridinium salt of HS is formed and freeze-dried. It is        dissolved and heated in a solution of DMSO/MeOH (9/1, v/v) for        various times at various temperatures e.g. 75 degrees C. for 6,        12, 24 (could be chosen at random or pre-determined by        experiment). Aliquots are removed (or alternatively, discrete        reactions can be carried out for the desired time points in        discrete locations) at desired time points, cooled, the pH        adjusted to ca. 8 (NaOH(aq)), precipitated into ethanol (cold),        filtered and washed (EtOH), then dialysed against distilled        water.        (iii). Partially re N-acetylate the HS.

The product (e.g. 25 mg) is dissolved in sat. aq. NaHCO₃ (1 ml), aceticanhydride added (in a number of known, varying quantities correspondingto known molar equivalents, depending on the extent required) at 4degrees C. and stirred for 1 hour. The cooling is removed and thereaction allowed to stir at room temperature overnight. The products areprecipitated into cold EtOH, filtered, washed (EtOH) and dialysedagainst distilled water.

(iv). Replace N-sulfates.

The products are dissolved in saturated aqueous NaHCO₃ andtrimethylamine.sulfurtrioxide complex added (in 10-fold molar excess, orgreater, if complete re N-sulfation is required) at 50 degrees C.,stirred for 24 hours, cooled and precipitated into EtOH (cold),filtered, washed (EtOH) and dialysed against distilled water.

(v). Degrade to oligosaccharides by heparitinase enzymes (could also usenitrous acid degradation or free radical degradation as well).

The polysaccharide (<1 mg/ml) is dissolved in the appropriate enzymebuffer (Ca(OAc)₂, NaOAc) and digestion carried out with the appropriateenzyme (e.g. heparitinase III, 1 ul per ml of polysaccharide solution,2.5 mU/10 ul), incubated at 37 degrees C. for the desired time or times.The enzyme digestion is stopped by briefly heating the samples at 100degrees C. (2-5 minutes).

(vi). Separate the oligosaccharides so formed into discreet physicallocations e.g. by strong anion exchange hplc, or electrophoresis.

The products are assayed singly, or in groups for a particular activity(or property) of interest. If required, something is ascertained abouttheir structure, for example, by disaccharide compositional analysis.

NB. At the end of each step (especially (ii), (iii) and (iv)) structuralelucidation (e.g. by NMR) may be required to check that the desiredlevel and type of modification has been successfully carried out. Afterstep (v), it may be required to check the degree of degradation e.g. byelectrophoresis or hplc.

(vii). Tuning method e.g. “analytical”.

It may be that, for instance, at step (vi), a particular structure fromthe diverse library is found to be active and this turns out to be rich,for example, in N-acetylated glucosamine, glucosamine 6-sulfate andiduronate 2-sulfate, as found by some structural elucidation method(e.g. disaccharide compositional analysis). It would therefore berequired to make a polysaccharide rich in these structures, which couldbe done as follows:

-   -   I(i) *de-N-sulfation    -   The pyridinium salt is formed and freeze-dried. This is        dissolved in a solution of DMSO/MeOH (9/1, v/v) and heated for 2        hours at 55 degrees C. Aliquots are removed at desired time        points, cool, the pH adjusted to ca. 8 (NaOH(aq)), precipitated        into ethanol (cold), filtered and washed (EtOH), then dialysed.    -   I(ii) *re N-acetylation    -   The product is dissolved in saturated aqueous NaHCO₃, add acetic        anhydride added (in 10-fold molar excess) at 4 degrees C. and        stirred for 1 hour. The cooling is removed and stirred at room        temperature overnight. The products are precipitated into cold        EtOH, filtered, washed (EtOH) and dialysed.    -   I(iii) ascertain overall degree of modification.    -   Following modification, the structural integrity of the        polysaccharide is checked (e.g. by NMR, in which the peaks        apparent in the spectra are correlated with the structures        present (averaged over the whole sample): This information can        be used to evaluate the degree of sulfation and acetylation at        the various positions within the sample).    -   *The products are then degraded by enzymes to the desired extent        (this can be tested first if required, but is a parameter that        can itself be tuned, for example, to generate more longer        fragments or more shorter fragments, as required).    -   I(iv) Degrade to oligosaccharides by heparitinase enzymes (could        also use nitrous acid degradation or free radical degradation as        adjuncts and/or alternatives).    -   The polysaccharide is dissolved (<1 mg/ml) in the appropriate        enzyme buffer (Ca(OAc)₂, NaOAc) and digestion carried out with        the appropriate enzyme added (e.g. heparitinase III of activity        2.5 mU per 10 ul, 1 ul per ml of polysaccharide solution),        incubating at 37 degrees C. for the desired time or times. The        enzyme digestion is stopped by briefly heating the samples at        100 degrees C. (5-10 minutes).    -   I(v) separate    -   The products are separated into discreet locations (for example,        by hplc).    -   The activity is checked and the structure of the most        interesting component(s) determined. If further adjustment of        the parameters is required, this is done to create further        libraries until satisfied that the activity (or whatever        property is of interest) has been optimised. This is an example        of the analytical tuning process described in FIG. 1.    -   N.B. An alternative tuning process is also available, which we        term the empirical tuning process and is described in FIG. 2. It        starts with selection of a product with a desired activity,        whose synthetic history is known, but whose structure may or may        not be. This process differs from the empirical tuning method at        points marked * in this example, where conditions can be varied        to give a range of similar, but distinct products and no        structural check need necessarily be made on the products.        Products are identified only by their separation characteristics        and/or activity. The former can be considered as providing no        information, i.e. it could be effectively ignored or,        alternatively, it could be considered to provide sketchy or        fuzzy information about structure e.g. more sulfated saccharides        tend to elute later from hplc columns than less sulfated ones,        but this does not provide a detailed description of its        structure    -   In both tuning processes, it is also possible to optimise the        ratio of two parameters, either structural and/or functional.

The result of these processes will be components with optimisedparameters of interest.

Example 6 Production of Diverse Library Components Containing ActiveFragments and Illustration that Tuning can Involve DegradationTechniques as Well as, or Instead of, Chemical Modifications

1. A sample of heparin is taken

2. Modifications to remove O-sulfates at positions glucosamine-6 andiduronate-2 to a range of extents is carried out, this also removes allN-sulfates at the same time.

The pyridinium salt is formed and freeze-dried. This is dissolved in asolution of DMSO/MeOH (9/1, v/v) and heated for a variety of time pointsat one temperature (or various temperatues as required). Aliquots areremoved at desired time points, (or alternatively, reactions are carriedout in discrete vessels for the required range of conditions) cooled,the pH adjusted to ca. 8 (NaOH(aq)), precipitated into ethanol (cold),filtered and washed (EtOH), then dialysed. The extent of modification isascertained e.g. by NMR. This forms a number of products with varyingdegrees of O-sulfation at position-2 of iduronate and position-6 ofglucosamine.

3. replace some N-acetyl groups and the remaining free amines withN-sulfate to give products with variable N-acetyl/N-sulfate ratios.

The product is dissolved in saturated aqueous NaHCO₃, acetic anhydrideadded (in a number of known, varying quantities) at 4 degrees C. andstirred for 1 hour. The cooling is removed and allowed to stir at roomtemperature overnight. The products are precipitated into cold EtOH,filtered, washed (EtOH) and dialysed. The extent of modification isascertained e.g. by NMR. This forms a number of products with varyinglevels of N-acetylation.

The remaining free amino groups are re N-sulfated by dissolving theproduct in saturated NaHCO₃, excess trimethylamine.sulfurtrioxide addedand the reaction heated at 55 degrees C. overnight. The reaction iscooled, precipitated into ethanol, filtered and dialysed againstdistilled water. The extent of modification in each component of thelibrary e.g. by NMR is ascertained. This yields a library of modifiedpolysaccharides containing variable O-sulfation at position 2- ofiduronate, position-6 of glucosamine and at the amine group ofglucosamine.

4. having produced this polysaccharide library, generate mixtures ofoligosaccharide fragments by partial enzymatic and/or chemical digestion

The sample is dissolved in lyase buffer (Ca(OAc)₂NaOAc) at <1 mg/ml andadd (e.g. 1 ul of heparitinase III enzyme per ml of polysaccharide)added and incubated at 37 degrees C. for various times. The progress ofdigestion can be monitored by removing aliquots at various time points,heating the samples briefly at 100 degrees C. and monitoring the extentof degradation e.g. by running the samples on an electrophoresis gel anddetecting the oligosaccharides (against standards) by staining with e.g.Alcian blue/Azure A.

5. separate these pools of mixed oligosaccharides e.g. by hplc and assayfractions for a particular activity of interest

A sample of the digestion (e.g. 0.5 mg in 1 ml water) is added toastriong anion exchange column and eluted with a linear gradient of NaCl(0-2M, pH 7, over 120 minutes at 1 ml per minute) monitoring the elutionposition of products by their absorbance at 232 nm. The eluant isfractionated into 1 ml tubes (e.g. at 1 ml/min). Samples can be assayedfor a particular activity of interest.

6. isolate oligosaccharide of interest and determine structural details

This process may yield, for example, a saccharide, which upon structuralelucidation e.g. by gel or mass spectrometry based sequencingtechniques, is revealed to be, for instance, a tetrasaccharidecontaining glucosamine N-sulfate groups, low levels of O-sulfatediduronate and sulfation at position-6 of glucosamine).

7. prepare a polysaccharide with very low levels of iduronate-2 sulfateand glucosamine-6 sulfate by,

-   -   I(i) A sample of HS is taken    -   I(ii) It is subjected to de-O-sulfation for a prolonged period        (also achieving de N-sulfation at the same time)    -   The pyridinium salt is formed and freeze-dried. This is        suspended in a solution of DMSO/MeOH (9/1, v/v) and heated for        24 hours at 100 degrees The sample is removed cooled, the pH        adjusted to to ca. 8 (NaOH(aq)), and the products precipitated        into ethanol (cold), filtered and washed (EtOH), then dialyses.        The degree of modification is ascertained e.g. by NMR.    -   I(iii) re-N-sulfate the remaining free-amino groups to        completion    -   The product is dissolved in saturated aqueous NaHCO₃, excess        trimethylamine.sulfurtrioxide added at 55 degrees C. and stirred        overnight. The reaction is cooled and the products are        precipitated into cold EtOH, filtered, washed (EtOH) and        dialysed. The degree of modification is ascertained e.g. by NMR.    -   I(iv) In a trial run, digest it extensively with the same enzyme    -   The sample is dissolved in lyase buffer (Ca(OAc)₂/NaOAc) at <1        mg/ml and enzyme (e.g. 1 ul of heparitinase III enzyme per ml of        polysaccharide) added. The digest is incubated at 37 degrees C.        for a variety of time points. The progress of digestion is        monitored by removing aliquots at various time points, heating        the samples briefly at 100 degrees C. checking (e.g. by        comparing the migration of the digested fractions against        standards by gel electrophoresis and staining with alcian        blue/azure A) for the degree of degradation achieved, until a        high yield of (in this case) tetrasaccharides has been obtained.        Digest more of the sample in the same way to obtain a large        quantity of pooled tetrasaccharides.    -   I(v) These pooled oligosaccharides are separated (e.g. by hplc)        and the fraction that contains the oligosaccharide of interest        is identified. This could be done on the basis of some        structural test (e.g. by mass spectrometry, sequence analysis,        elution position on hplc) and/or some functional property. This        illustrates that not only the chemical modification steps but        also the enzymatic step is a tunable aspect of the process.

Example 8 An Illustration of the Generation of Structurally DiverseOligosaccharide Libraries

(a). Generation of Diverse HS Analogue Libraries.

The generation of diverse HS analogue libraries from a heterogeneouspolysaccharide is illustrated in FIG. 6. The electrophoresis profile ofa partial digestion with heparitinase II of a structurally diversepolysaccharide is shown (panel A). The products are first fractionatedon the basis of their hydrodynamic volume on Sephadex G-50 (panel B).This profile is similar to that obtained from a typical enzymaticdigestion of heparin or heparan sulfate. However, when peakscorresponding to particular hydrodynamic volume ranges, in this caseDP12 of bovine lung heparin derived standards, are further fractionatedon the basis of overall charge by HPAEC (panel C), a distinct pattern isobserved. Instead of a range of separable peaks, typical of a modestnumber of saccharides, the overall chromatogram is bound by anapproximately Gaussian envelope, inside of which are discrete, regularlyspaced peaks. This is a typical example of the appearance of HPAEchromatograms of gel chromatography fractions from enzyme digestions ofthis kind of highly heterogeneous polysaccharide. The heterogeneity ofeach of these peaks is further demonstrated by their profile on anelectrophoresis gel (examples labelled A, B and C in FIG. 5 (panel D))which separates them on the basis of a combination of charge, size andconformation. Comparing the appearance of these diffuse bands (which,because of their lower overall sulfation levels, run higher up the gel),with their more highly charged and homogeneous counterparts derived frombovine lung heparin (shown as standards, S in panel D), it is clear thatthe standards run as tighter bands and this is especially evident forthose larger than DP 6. Each of the discrete peaks on the HPAEC tracecontains a diverse range of structures forming sub-libraries ofoligosaccharides. These data together with the composition analysis ofpeaks A, B and C from HPAEC (panel E) suggests that they contain complexmixtures of oligosaccharides.

(b). The Use of the Library to Select Active Structures ApproachingMinimum Complexity

An illustration of the use of the library to select activeoligosaccharide sets (or sub-libraries) with minimum size and charge isshown in FIG. 6 for the fibroblast growth factor-receptor (FGF/FGFR)system in an in vitro cell assay with Baf3 cells, in which the abilityof fractions to support signalling with FGF-1/R2c is measured. Testingthe activities of fractions from the partial heparitinase digestionseparated by gel chromatography (panel A) allows a pool ofoligosaccharides to be selected on the basis of activity whileminimising size and charge. It is noteworthy that higher hydrodynamicvolume does not necessarily bestow higher activity, as illustrated inFIG. 6 (panel A) for three fractions denoted B, D and I. Furtherseparation, on the basis of charge of the smallest significantly activefraction, in this case D, by HPAEC and subsequent testing of theresultant fractions for activity, allows the search to be focussed.Higher charge does not necessarily correlate with higher activity asillustrated by the activities of HPAE fractions c, f, l and r (panel B).Fractions exhibiting both higher activity (e.g. f) and lower activity(e.g. r) than the parent (D) can be identified, indicating that a degreeof specificity is present in FGF/FGFR/HS interactions. It should also benoted that (polymeric) heparin, which is used here as a positivecontrol, is likely to appear a disproportionately effective activatorcompared to oligosaccharides because it possesses many more activesites. Additional iterations of the separation and screening processwill allow increasingly focussed structure/activity relationships to besought.

(c) Methods

1. Chemical Preparation of Heterogeneous Polysaccharide

(a) Partially De O-, Completely De N-Sulfated Heparin

Porcine intestinal mucosal heparin (Celsus Labs, Cincinatti, Ohio, USA,5 g) was converted to the pyridinium salt by passage through Dowex W-50cation exchange resin (H⁺ form), neutralised with pyridine andfreeze-dried (4.9 g). This was then suspended in a solution ofDMSO/MeOH, 9/1, v/v (100 ml) and heated at 80° C. for a time (24 h),determined empirically following removal of aliquots (10 ml), recoveryand analysis by NMR. The product was recovered and purified by gelchromatography and analysed by NMR to verify its structuralheterogeneity in terms of partial de O-sulfation and complete deN-sulfation. It was then subjected to partial re N-acetylation.

(b) Partial Re N-Acetylation

Partial re N-acetylation was achieved with acetic anhydride in asaturated solution of sodium bicarbonate upon the partially deO-sulfated polysaccharide but its extent, determined empirically bymonitoring aliquots by NMR following recovery, was limited bycontrolling the quantity of acetic anhydride used. Products wereisolated and characterised by NMR and, following exhaustive degradationwith heparitinase enzymes, disaccharide analysis.

(c) Re N-Sulfation of Remaining Unsubstituted Amino Groups

The remaining free-amino groups were re N-sulfated (twice) usingtrimethylamine sulfurtrioxide as the sulphating agent. Following thisprocedure, the compound was purified by gel chromatography and its highlevels of heterogeneity confirmed by compositional analysis: UA-GlcNAc;24.5%, UA-GlcNAc(6S); 13.7%, UA-GlcNS; 7.0%, UA-GlcNS(6S); 13.0%,UA(2S)-GlcNS; 13.7%, UA(2S)-GlcN(6S); 13.6%, UA(2S)-GlcNSAc; 11.4%,UA(2S)-GlcNAc(6S); 3.1%.

2. Characterisation of Polysaccharide

(i) NMR: The effectiveness of the chemical treatments were monitored by¹H and ¹³C NMR spectroscopy at 500 and 125 MHz in D₂O on a Brukerspectrometer operating at 27° C. Chemical shifts (relative to anexternal standard) were assigned and the compound characterised by NMR.

(ii) Disaccharide analysis following exhaustive digestion withheparitinases I, II and III: Samples (typically 100 ug) wereexhaustively digested with a combination of heparitinase enzymes I, IIand II (Seikagaku) in lyase buffer at 37° C. (500 mM NaOAc, 2.5 mMCa(OAc)₂, pH 7). Subsequent comparison with disaccharide standardsfollowing separation by HPAEC on a Propac PA-1 column (4×250 mm, 0-2 MNaCl gradient over 90 mins, detecting at 232 nm) allowed each componentto be quantified.

3. Partial Degradation of Heterogeneous Polysaccharide with HeparitinaseII

The polysaccharide (50 mg) was partially digested with hepaitinase II(Seikagaku) in lyase buffer (as above) at 37° C. The progress of thedigestion was monitored by electrophoresis of the products by stainingwith Alcian blue/Azure A and was stopped when a range of digestedproducts was detected with reference to a pair-wise ladder of heparinfragments.

4. Fractionation of Products by Gel Permeation Chromatography

The partially digested products were separated on the basis of theirhydrodynamic volume on a column of Sephadex G-50 (2.5 cm×1.75 m) elutingwith 100 mM NH₄HCO₃, detecting at 232 nm. The column was calibrated(before and after separation) with a pair-wise ladder of heparinoligosaccharides derived by partial heparitinase digestion. Fractions(denoted A-O) were desalted, quantified (A₂₃₂) and tested for efficacyin a number of assays following quantification.

5. Fractionation of Hydrodynamic Volume Defined Products by HPAEC

Selected fractions from the gel permeation chromatography separationwere desalted and fractionated on HPAEC on a Propac PA-1 column (4×250mm, 0-2 M NaCl gradient over 90 mins, detecting at 232 nm). Peaks werecollected (selected peaks were denoted A, B and C for use in theexperiments shown in FIG. 5, but the full range were denoted a-p for usein those shown in FIG. 6) de-salted and quantified (A₂₃₂) for subsequentanalysis and testing.

6. BaF3 Cell Assay with FGFs and FGFRs.

BaF3 cells transfected with the appropriate receptor were maintained inRPMI-1640 supplemented with 10% foetal calf serum, 2 mM L-glutamine,1000 U·ml⁻¹ pen G, 50 μg·ml⁻¹ streptomycin sulfate and 2 ng·ml⁻¹ IL-3.Assays for saccharide function were as follows. Briefly, BaF3 cells weretransferred to 96 well plates at 10000 cells per well in 100 μl mediumwithout IL-3, supplemented with 1 nM of the appropriate FGF saccharidesamples. Pools of saccharides from gel chromatography (denoted A to O)were used between 1.0 ng·ml⁻¹ and 10,000 ng·ml⁻¹ and from HPAEC (denoteda to t) were used between 0.1 and 3,000 nM. Cells were incubated (37°C., 72 hours). 5 μl MTT (5 mg·ml⁻¹ in PBS) was added and cells incubated(a further 4 hours, 37° C.). Cells were solubilized (10% SDS, 0.1 NHCl). Absorbance of solubilized samples was measured at 570 nm.

1. A method for the production of a library of heparan sulfatederivatives said method comprising a combination of chemicalmodification steps in which at least one, two or three modificationsteps of said combination are selected from the group A to O wherein: A.partial de N-sulfation in glucosamine B. complete de N-sulfation inglucosamine C. partial de N-acetylation in glucosamine D. complete deN-acetylation in glucosamine E. re N-sulfation in glucosamine of allavailable amino groups F. re N-acetylation in glucosamine of allavailable amino groups G. partial re N-sulfation in glucosamine H.partial re N-acetylation in glucosamine I. complete de-O-sulfation atposition 6 of glucosamine J. partial de-O-sulfation at position 6 ofglucosamine K. partial de-O-sulfation at both position 6 of glucosamineand 2 of iduronate accompanied by complete de N-sulfation inglucosamine. L. complete de-O-sulfation at both position 6 ofglucosamine, 2 of iduronate and de-N-sulfation in glucosamine M. partialde-O-sulfation at position 6 and complete de-N-sulfation of glucosamineN. complete de-O-sulfation at position 2 of iduronate O. completede-O-sulfation at position 6 and de N-sulfation of glucosamine andpartial de-O-sulfation of iduronate.
 2. The method of claim 1 whereinall steps of said combination are chosen from the group A to O.
 3. Themethod according to claim 1 wherein said library is structurally morediverse than the heparan sulfate starting material from which it isderived.
 4. The method according to claim 1 wherein at least onemodification step in said combination is a partial modification.
 5. Themethod according to claim 1 wherein at least one complete or partialmodification is carried out at the amino function (N—) of glucosamine.6. The method according to claim 1 wherein at least two modificationsteps in said combination are partial modifications.
 7. The methodaccording to claim 1 wherein at least three modification steps in saidcombination are partial modifications.
 8. The method according to claim1 wherein a first step of modification is chosen from A, B, C or D, suchthat wherein step A is chosen, optional subsequent steps are one or moreof E, F, G, H, I, J, K, L, M, N, O, in any combination, or wherein stepB is chosen, optional simultaneous or subsequent steps are one or moreof E, F, G, H, I, J, K, L, M, N, O, in any combination.
 9. The methodaccording to claim 8 wherein a second step of modification chosen fromE, F, G, or H is performed upon the modified products of said firststep.
 10. The method according to claim 9 wherein a third step ofmodification chosen from A, B, C, D, E, F, G, H, I, J, K, L, M, N, O isperformed upon the modified products of said second step.
 11. The methodaccording to claim 10 wherein a fourth step of modification chosen fromA, B, C, D, E, F, G, H, I, J, K, L, M, N, O is performed upon themodified products of said third step.
 12. The method according to claim11 wherein the combination of modifications is chosen from a first stepand second to fourth optional steps such that: Optional OptionalOptional First Step Second Step Third Step Fourth Step B(+/−any of I toO) G F/H B(+/−any of I to O) H E/G B(+/−any of I to O) E B(+/−any of Ito O) F A F +/−any of I to O E/G A H +/−any of I to O E/G


13. The method according to claim 12 wherein said first stepmodification is B (+/− any of I to O), said second step modification isH, and said third step modification is E or G.
 14. The method accordingto claim 12 wherein said first step modification is B (+/− any of I toO), said second step modification is G, and said third step modificationis F or H.
 15. The method according to claim 1, the method comprisingthe additional steps (singly or jointly) of (a)(i) determining at leastone functional property of one or more compounds; (b)(i) making afurther library via the method according to claim 1 wherein saidmodifications are chosen according to the functional determination ordeterminations made in step (a)(i); and/or; (a)(ii) determining at leastone structural feature of one or more compounds; (b)(ii) making afurther library via the method according to claim 1, wherein saidmodifications are chosen according to the structural determination ordeterminations made in step (a)(ii); and/or, (b)(iii) making a furtherlibrary via the method according to claim 1, wherein said modificationsare chosen according to both said functional determination(s) made instep (a)(i) and said structural determination(s) made in step (a)(ii).16. A method of producing a supplementary library of modified heparinderivatives comprising steps (singly or jointly) (i) screening a libraryof heparan sulfate derivatives for compounds which have particularstructural and/or functional characteristics, (ii) determining at leastone structural feature of the compounds having said particularstructural and/or functional characteristics, or (iii) determining atleast one functional property of the compounds having said particularstructural and/or functional characteristics, or (iv) determining atleast one functional and one structural property of the compounds havingsaid particular structural and/or functional characteristics; steps(ii), (iii) and (iv) being followed by step (v) making said furtherlibrary via the methods of claim 1 wherein the modifications and numberof modification steps are chosen according to the determinations ofsteps (ii), (iii) or (iv).
 17. The method according to claim 16 whereinthe library of step (i) is made by a method according to claim
 1. 18.The method according to claim 16 wherein at step (v) a singlecombination of modification steps is chosen in order to reproduce onlythe compound(s) having said desired characteristics.
 19. The methodaccording to claim 16 wherein the structural determination(s) made atstep (ii) or (iv) is/are provided by the discreet known location, in aspatially separated library, of the compounds having said particularstructural and/or functional characteristics.
 20. A library containingat least two heparan sulfate derivatives produced by the method ofclaim
 1. 21. The library according to claim 20 in which the compoundscontained therein are spatially separated from each other.
 22. Thelibrary, or components of the library produced by the method of claim 1,in which said components are: (a) spatially separated, (b) spatiallyseparated into defined locations, (c) spatially separated into definedlocations and attached to a surface, (d) spatially separated such thatan interaction between one or more compounds within said library and themolecule, complex of molecules, cell or organism of interest can bedetected, (e) spatially separated into defined locations such that aninteraction between one or more compounds within said library and themolecule, complex of molecules, cell or organism of interest can bedetected, (f) spatially separated into defined locations and attached toa surface such that an interaction between one or more compounds withinsaid library and the molecule, complex of molecules, cell or organism ofinterest can be detected.